Carlos Angulo

Food-Grade Organisms as Vaccine Biofactories and Oral Delivery Vehicles

The use of food-grade organisms as recombinant vaccine expression hosts and delivery vehicles has been explored during the past 25 years, opening new avenues for vaccinology. Considering that oral immunization is a beneficial approach in terms of costs, patient comfort, and protection of mucosal tissues, the use of food-grade organisms can lead to highly advantageous vaccines in terms of costs, easy administration, and safety. The organisms currently used for this purpose are bacteria (Lactobacillus and Bacillus), yeasts, algae, plants, and insect species. Herein, a comparative and updated scenario on the production of oral vaccines in food-grade organisms is provided and placed in perspective. The status of clinical evaluations and the adoption of this technology by the industry are highlighted.

Dietary administration of microalgae alone or supplemented with Lactobacillus sakei affects immune response and intestinal morphology of Pacific red snapper (Lutjanus peru).

The aim of this study was to evaluate the effect of dietary microalgae Navicula sp single or in combination with Lactobacillus sakei on growth performance, humoral immune parameters and intestinal morphology in Pacific red snapper, Lutjanus peru. The experimental fish were grouped into four treatment diets which were a control diet (commercial diet, Control), silage microalgae Navicula sp plus L. sakei (10(6) CFU g(-1), Navicula + L. sakei), lyophilized microalgae (Navicula) and L. sakei (10(6) CFU g(-1), L. sakei). The blood and intestine samples were collected on week 4 and 8. The weight gain showed an additive effect of Navicula + L. sakei at 8 weeks of treatment compared with fish fed control diet. Overall, physiological parameters such as total protein and hemoglobin were increased in fish fed with Navicula and L. sakei diets at 4 and 8 weeks of feeding assay, respectively. There was a significant improvement in immune parameters, principally in myeloperoxidase, lysozyme, total antiproteases activities and IgM in fish fed with Navicula + L. sakei and L. sakei diets at 4 or 8 weeks of treatments. Serum antioxidant capabilities revealed significant increase in phosphatase alkaline, esterase, protease, superoxide dismutase and catalase in groups which received diet supplemented with Navicula + L. sakei and L. sakei diets. Finally, light microscopy observations revealed no effect of experimental diets on microvilli height. Curiously, the presence of vacuoles inside the enterocytes was significant higher in the intestine of L. sakei group after four or six weeks of feeding. Elevated intraepithelial leucocyte levels and melanomacrophages centers were observed in fish fed Navicula or control diets at any time of the experiment. To conclude, the results of the present study demonstrate that the fish that were fed with Navicula + L. sakei or L. sakei diets yielded significantly better immune status and antioxidant capabilities.

Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines

Chikungunya virus is an emerging pathogen initially found in East Africa and currently spread into the Indian Ocean Islands, many regions of South East Asia, and in the Americas. No licensed vaccines against this eminent pathogen are available and thus intensive research in this field is a priority. This review presents the current scenario on the developments of Chikungunya virus vaccines and identifies the use of genetic engineered plants to develop attractive vaccines. The possible avenues to develop plant-made vaccines with distinct antigenic designs and expression modalities are identified and discussed considering current trends in the field.

Molecular cloning and comparative responses of Toll-like receptor 22 following ligands stimulation and parasitic infection in yellowtail (Seriola lalandi).

TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. In this study, we cloned the full-length cDNA sequence of yellowtail (Seriola lalandi) TLR22 (SlTLR22). The complete cDNA sequence of SlTLR22 was 4208 bp and encodes a polypeptide of 961 amino acids. Analysis of the deduced amino acid sequence indicated that SlTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 91-633) and one C-terminal LRR domain (LRR-CT, residues 693-744) in the extracellular region, and a TIR domain (residues 800-943) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced SlTLR22 has the highest sequence identity to turbot TLR22 (76%). Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SlTLR22 mRNA in all examined tissues with higher levels in the head kidney, intestine, skin and spleen. Further, SlTLR22 expression was significantly up-regulated following TLR ligands injection with lipopolysaccharide (LPS), CpG ODN2006 and polyinosinic: polycytidylic acid (poly I:C) in spleen and liver. Amyloodinium ocellatum infection also induced a high expression of SlTLR22 in spleen, intestine, muscle, skin and gill, with maximum increases ranging from 1000 to 100 fold upon different ligands and organs. Finally, histological examination in gill tissue confirmed infection by the parasite and histopathological lesion was observed also in spleen and skin. These findings suggest a possible role of SlTLR22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and parasites.

Humoral immune response and TLR9 gene expression in Pacific red snapper (Lutjanus peru) experimentally exposed to Aeromonas veronii.

Aquaculture production of Pacific red snapper Lutjanus peru is growing rapidly in Mexico, especially in Gulf of California. As it is a relatively new aquaculture species there are few reports evaluating its immune response to pathogens. The Gram-negative bacteria Aeromonas veronii is a heterogeneous organism that causes the disease known as motile aeromonad septicemia, which is responsible for serious economic loss in seabream culture due to bacterial infections. For the purpose of this study, juvenile Pacific red snapper specimens were intraperitoneally injected with low doses of A. veronii (1 × 10(6) CFU ml(-1)). Changes in humoral immune parameters (total protein, myeloperoxidase, lisozyme and antiprotease activities and IgM levels), as well as superoxide dismutase and catalase activities, and TLR9 gene expression were evaluated 24 and 48 h after injection. Overall, the results showed an enhanced in humoral immune parameters and SOD and CAT activities in fish infected with A. veronii compared with control group at 24 or 48 h. By real time PCR assays, the basal mRNA transcripts of TLR9 showed that were highly expressed in intestine and leucocytes compared to skin, head kidney, liver and gill. Then, the mRNA expression levels of TLR9 in head kidney, skin, liver and intestine were analyzed in non-infected and experimentally infected fish 24 and 48 h after injection. A. veronii up-regulated the expression of TLR9 at 24 or 48 h of exposure in all samples analyzed except in liver. Interestingly, intestine produced the greatest increase in transcript levels upon exposure (48 h) to A. veronii. Taken together, our results suggest that low doses of A. veronii infection inducing humoral immune system and TLR9 immune gene in Pacific red snapper that can be useful in the health control of this species.

Bacillus subtilis comes of age as a vaccine production host and delivery vehicle

Bacillus subtilis is a vaccine production host and delivery vector with several advantages such as a low production cost, straightforward administration as it is safe for human consumption and the production of spores exerting adjuvant effects. This review summarizes the expression approaches and provides an updated outlook of how a myriad of pathogens have been targeted under this technology. Furthermore, by reviewing the literature, several promising candidates in terms of immunogenic and immunoprotective potential have been identified. The immune profiles achieved comprise either humoral or cellular responses, which reflect versatility for application in the fight of distinct pathologies that demand specific polarization on the immune responses. Some perspectives for this field are also envisioned.

A novel design of a multi-antigenic, multistage and multi-epitope vaccine against Helicobacter pylori: An in silico approach

Helicobacter pylori have colonized the gastric mucosa of half of the population worldwide. This bacterium is classified as a definitive type I carcinogen by the World Health Organization and no effective vaccine has been found against it yet. Thus, a logical and rational vaccine design against H. pylori is necessary. Because of its tremendous complexity and elicited immune responses, the vaccine design should considered multiple antigens to enhance immune-protection, involved in the different stages of pathogenesis besides inducing a specific immune response by B- and T-cell multi-epitopes. In this study, emphasis was placed on the design of a new unique vaccine named CTB-multiHp. In silico techniques were used to design a chimeric construct consisting of cholera toxin B subunit fused to multi-epitope of urease B (residue 148-158, 188-198), cytotoxin-associated gene A (residue 584-602), neutrophil activating protein (residue 4-28), vacuolating cytotoxin gene A (residue 63-81), H. pylori adhesine A (residue77-99), heat shock protein A (residue 32-54) and gamma glutamyl transpeptidase (residue 271-293). The tertiary structure and features of the vaccine were analyzed. The chimeric protein was expressed in Escherichia coli BL21 and the serology analyses indicated that the CTB-multiHp protein produced exhibit immune-reactivity. The results showed that CTB-multiHp could be a good vaccine candidate against H. pylori. Ongoing studies will evaluate the effects of CTB-multiHp against H. pylori infection.

In silico epitope analysis of unique and membrane associated proteins from mycobacterium avium subsp. paratuberculosis for immunogenicity and vaccine evaluation

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis disease affecting ruminants worldwide. The aim of this study was to identify potential candidate antigens and epitopes by bio and immuno-informatic tools which could be later evaluated as vaccines and/or diagnosis. 110 protein sequences were selected from MAP K-10 genome database: 48 classified as putative enzymes involved in surface polysaccharide and lipopolysaccharide synthesis, as membrane associated and secreted proteins, 32 as conserved membrane proteins, and 30 as absent from other mycobacterial genomes. These 110 proteins were preliminary screened for Major Histocompatibility Complex (MHC) class II affinity and promiscuity using ProPred program. In addition, subcellular localization and host protein homology was analyzed. From these analyses, 23 MAP proteins were selected for a more accurate inmunoinformatic analysis (i.e. T cell and B cell epitopes analysis) and for homology with mycobacterial proteins. Finally, eleven MAP proteins were identified as potential candidates for further immunogenic evaluation: six proteins (MAP0228c, MAP1239c, MAP2232, MAP3080, MAP3131 and MAP3890) were identified as presenting potential T cell epitopes, while 5 selected proteins (MAP0232c, MAP1240c, MAP1738, MAP2239 and MAP3641c) harbored a large numbers of epitopes predicted to induce both cell- and antibody-mediated immune responses. Moreover, immunogenicity of selected epitopes from MAP1239c were evaluated in IFN-γ release assay. In summary, eleven M. avium subsp. paratuberculosis proteins were identified by in silico analysis and need to be further evaluated for their immunodiagnostic and vaccine potential in field and mice model.

Debaryomyces hansenii up regulates superoxide dismutase gene expression and enhances the immune response and survival in Pacific red snapper (Lutjanus peru) leukocytes after Vibrio parahaemolyticus infection

ABSTRACT Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non‐pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head‐kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head‐kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head‐kidney and spleen leukocytes. HighlightsMn‐SOD, icCU/Zn‐SOD and catalase genes were identified and characterized in Pacific red snapper.D. hansenii stimulate antioxidant immune‐related gene expression.D. hansenii protects leucocytes against the pathological damages of V. parahaemolyticus.D. hansenii increased phagocytosis activity and cell viability of fish leucocytes.D. hansenii reduced necrotic cell death provoked by V. parahaemolyticus.Antioxidant immune parameters are enhanced by D. hansenii.

Antioxidant screening and phenolic content of ethanol extracts of selected Baja California Peninsula macroalgae

The Baja California Peninsula in México has about 670 species of macroalgae along its coast. Species richness increases the probability of finding native macroalgae with potential as sources of bioactive compounds suitable for health, pharmacological, and cosmetic ingredients. To understand the biotechnological value of macroalgae from the peninsula, ethanol extracts from 17 macroalgae (four Chlorophyta, six Rhodophyta, seven Ochrophyta) were screened for antioxidant potential. To determine the antioxidant capacity of macroalgal extracts, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power, and nitric oxide radical scavenging as well as total phenolic content (TPC) were measured. Extracts of the brown macroalgae were most active. Among these, Eisenia arborea, Padina concrecens, and Cystoseira osmundacea had the highest TPC and exhibited the strongest radical scavenging activities. Correlations were found between TPC macroalgal and scavenging capacity, indicating an important role of polyphenols as antioxidants. This suggests that some brown macroalgae from Baja California Peninsula may be a good source of natural bioactive compounds.